Kohler and Milstein have provided a method for successful production of monoclonal antibodies, as reported in Nature, 256: 495-497 (1975). This method involves immunization of mice, followed by removal of the mice spleen cells, and fusion of the spleen cells with mouse myeloma cell lines, in a medium containing polyethylene glycol. Screening methods are then used to determine which, if any, of the resulting fused cells, or "hybridomas," produce monoclonal antibodies to a particular antigen.
This technique has not proven to be generally effective in producing hybridomas which, in turn, produce human monoclonal antibodies. As a result, several different approaches have been taken to improve production of hybridomas or other cell lines which produce human monoclonal antibodies.
One line of inquiry involved the fusion of immunized human B lymphocytes with mouse myeloma cell lines, an "interspecies" hybridization. A second line of inquiry involved "intraspecies" fusion, of immunized human B lymphocytes, with either human myeloma, or human lymphoblastoid cell lines. "Interspecies" hybridization allows the use of well-known mouse myeloma lines NS1 or SP2/oAG14. These cell lines offer the advantage of efficient fusion frequency, and high levels of immunoglobulin production, but present the serious problem of preferential loss of human chromosomes. The loss of human chromosomes results in instability of antibody production. Intraspecies hybridization has not been effective either. Fusion frequencies are low, often below 10.sup.-5, and antibody titer is also very low.
Another line of inquiry has been cell transformation by Epstein Barr Virus (EBV). In the EBV transformation method, human cells, especially B lymphocytes are immortalized by in vitro EBV infection. This method has been used to produce human antibodies to synthetic haptens, tetanus toxoid, diptheria toxoid, Rh antigens, influenza virus, human immunoglobulin complexes, Plasmodium falciparum antigens, acetylcholine receptor, pneumococci, and cytomegalovirus.
EPV transformation allows development of cell lines which produce a multitude of desired antibodies. Additionally, this method opens the possiblity of expanding the number and range of immune B lymphocytes useful in hybridization. EBV transformed cell lines, when hybridized to human or to mouse myeloma cell lines, give generally higher levels of antibody production than the original lymphobliastoid line.
EBV techniques are of limited use, however. At best, EBV tarnsformed cells have produced low titers of antibody, generally between 1 and 100 ng/ml. Additionally, uncloned lymphoblastoid cells have contributed to instability of antibody production.
The reason why the antibody secretion of lymphoblastoid cells is unstable over time must be seen in the heterogeneity of the fledging cell lines, which results in overgrowth of the culture by unwanted cells. Thus, EBV transformed cell lines nearly always contain cell mixtures with individual growth kinetics. Any change in the heterogeneous mixture, such as a slight acceleration of the growth cycle of the unwanted cell type, leads to domination of the culture by that type of cell, at the expense of the desired clone.
Early cloning can be expected to alleviate this problem. The two methods traditionally used in cloning, i.e., limiting dilution in liquid culture, and colonial growth in semisolid media, however, do not apply to EBV transformed cell with the same efficiency as with, e.g., mouse hybridoma cells. Limited dilution cloning relies on diluting cells to the point where, statistically, there is only one seed cell per culture well, and each culture (or clone) growing from the cell can be regarded as pure for that cell. In laboratory work, it has been found that very few lymphoblastoid cell lines which produce specific antibodies can be adapted to this method.
Lymphoblastoid cells have been grown in liquid culture in direct contact with irradiated fibroblasts, plated on the bottom of culture plates but, heretofore when agarose cultures were used the fibroblasts were separated from the lymophoblastoids, or any other cells to be cloned, by an agarose layer. It is thought that fibroblasts create an environment which supports growth of cells by providing an extracellular matrix, or biomatrix, composed of several types of collagens and other proteins.
Growth in semisolid agarose medium relies upon dilution of cells to the point at which the colonies which grow from single cells suspended in agarose grow separately from each other, such that the colonies are physically distinct. When colonies are size distinguishable under stereo microscopes, they are picked by micropipette and transferred into liquid medium for further growth. The level of cloning efficiency, however, has never exceeded more than about 3% of input cells.
It has now been found that an improvement in the semi-solid agarose method of growing cell clones establishes conditions where much higher frequencies of cell cloning and growth may be obtained. The method comprises using two layers of agarose, where the top layer contains both fibroblast cells and cells sought to be grown. The lower layer of agarose, which is in contact with the upper layer, has suspended therein additional fibroblasts.
This two layer semi-solid agarose system provides an "environment" of feeder cells, i.e., the fibroblasts, which supports growth of the desired clones.